Menu

1964-1965 (Q)

[00:00:00] This is Book Q, begins on October 16th, Friday, 1964. On the left-hand side of this entry is a rather clear set of statements about what I think is happening — or I thought was happening with regard to the subunits of haptoglobin and various disulfide bonds which were possible. It’s quite a nice summary. So I was clearly getting to understand what the monomers and so on were like. On the right-hand page, which is page 1, I’m — got a completely new idea buzzing around in my head, obviously, because I’m thinking about ferredoxin, [00:01:00] which is a protein with a lot of iron, bound to multiple SH groups, as a little diagram showed. And, “This protein has partial duplication and should be useful for unequal crossover tests, since it’s easy to prepare in Clostridium pasteurianum.” That’s the beginning statement. And so I’m beginning to think about some tests of what might happen in — with unequal crossing over, using a protein — a different protein.

And so here, in the following pages, are what I was attempting to, and purifying the protein from the bacteria. Clear on page 3, for [00:02:00] example, that — various extracts of the cells and so on. And continuing on page 5, Monday, October 19, having a good staining method to detect the ferredoxin. Some benzidine reactions are working. And continuing that general theme, Tuesday, October 20th, and the following pages, all towards preparing the protein.

And then, on page 13 of this book, Thursday, October 24, I have a comment on “General plan for antibody transfer test.” Where this idea is coming from, I’m not cl‑‑ quite sure, looking back at this time, although I suspect it’s related to a [00:03:00] paper that I later published, on antibody viruses, an idea that antibodies might be coded for in structures which I call antibody viruses, which might be transferred between cells. The — that was published later, in July of 1965, under the title, “Antibody Induction and Tolerance.” “Antibody viruses may mediate the primary response and transform cell to give the secondary respon‑‑” It looks as if this page in the 1964 notebook, end of ’64, is presaging that one, which is published nearly a year later. Anyway, to — the idea is clearly stated that — to take some cow cells and some sheep cells [00:04:00] and see if it would be possible to make the splee‑‑ cow cells make antibody against sheep cells and vice — the cow cells make antibody against cow cell and the sheep cells make antibody, and sheep cell, etc., by transferring things. A little bit obscure but the idea is stated more clearly than I’ve spoken about it. But nothing immediately done. Because the following paper con‑‑ following page continues with purifying ferredoxin.

October 26, Monday, pages 19 and 18, some thoughts on how to get gradient for elution of gels. But I had seen earlier, in [00:05:00] looking at the books, a rather obscure method making a gradient for elution of proteins from columns.

Continuing to prepare ferredoxin, over the next few pages. Tests on October 29th, Thursday, page 23, a G‑100 column, attempting to elute with a gradient — presumably, but doesn’t actually say. So. Ferredoxin continues, in G‑50 instead of G‑100. “Fine spherical column, made like the others. Excellent characteristics.” And they’re giving three [00:06:00] partial — partially separated zones, page 27. Feeling rather pleased about it. “Continues to improve. Good separation of the largest. Next largest not so good,” etc. — trying to purify the protein, with gradient electro‑‑ elution, along a side-fractionating column. Continuing, November 2nd, page 31, repeat the test with G‑50. What about storage? — page 33.

And then back to preparing, as I have done for quite a — as I did for quite a long time, different Bence Jones proteins. Here again is a — and a new one, on Tuesday, November 10th, page [00:07:00] 35, 1.65 liters of urine from [name redacted] — Bence Jones protein. And the next few pages are devoted to purifying the protein.

I note, on pages 42 and 43, some of the handwriting there is different. And I think it — earlier in the book, I’d noticed the name Nancy, “Nancy did this” or so on. So this writing, presumably, is Nancy, very neat. But my writing is on the same [00:08:00] pages. I think Nancy must have been a technician. At this stage of my life, afraid I don’t remember who she was. Might be possible to find out. Pages 44 and 45 with her neat writing and my rather crude writing, “Q‑42, pooled,” in addition to what she had written. Test of the purified Bence Jones protein, on 47. Back to my writing again.

Ah, here we come. On Wednesday, November — I think — 25th, page 49, be‑‑ s‑‑ with a comment, “Haptoglobin splits again!” exclamation mar‑‑ still trying to understand how to get these polymers to dissociate into monomers and how to resynthesize them, which [00:09:00] turns out to be finally understood later on in this particular book, but not yet. Trying, on November 27th, and continuing on the next few pages. Room temperature, haptoglobin splits, on Friday, November 27. Rather ineffective. A rough test of slow-cool, etc., page 57. And so it goes on.

Then I see, on page 59, ag‑‑ Wednesday, December 2nd, that there is a pretty clear splitting in some of these samples. They’re labeled 1, 2, 3, 4, on page 58. And it’s [00:10:00] obvious that the sample 4 has — most of the polymers have gone — but leaving them… The comment is that “Resynthesis is clear.” So the gel was run again, to see what happened when they were left. Concerned about oxidation being a problem. Because I still have the idea that the disulfide bonds are being formed by oxidation. Samples from Q‑56, “Careful repeat. All stoppered” anaerob‑‑ “stoppered aerobically and left at room temperature,” to see what would happen. But obviously, concerned with it.

[00:11:00] Various techniques, on Wednesday, December 2nd, aimed at having control over whether there is oxygen in the system or not. Here is a nitrogen incubation test, on Saturday, December 5th. And everything (laughs) looks the same, at this point, wi‑‑ but the comment is, “Obviously, the gassing was only successful in some of the tubes.” The… More attempts [00:12:00] at oxidation versus temperature, again, and eq‑‑ and equilibrium, on Sunday, December 6, page 67, with samples set up with nitrogen, etc. — ice and room temperature and 37. The results being considered on the following page.

And then, on page 71 of this book, I have a DTDG test, dithiodiglycol, which I know, in the end, are going to be — to lead to the solution of the problem. But I’m not yet clear of this. But I [00:13:00] set up experiment with haptoglobin plus mercaptoethanol, at 7-millimolar, and haptoglobin plus 0.1-molar dithiodiglycol. And then added a trace of mercaptoethanol to the… This is the order of addition, is what’s being tested here. One starts with haptoglobin plus mercaptoethanol, at 7-millimolar, and then adding 0.1-molar dithiodiglycol, at 37. All at 37. And the other being, at 37, starting with haptoglobin plus dithiodiglycol and adding, then, mercaptoethanol after. This is — was their plan. [00:14:00] With a note that “This experiment will largely test rate, since no pre-incubations are made.”

However, on page — the following page, it’s very clear that things are happening with the dithiodiglycol, mercaptoethanol. Show a comment, “Very promising. But needs the thiol, to go.” In other words, the dithiodiglycol at 0.1-molar will help — will cleave the polymers but needs dithiodiglycol there to — the other way around — that the dithiodiglycol will encourage [00:15:00] splitting, if there is the thiol there as well. Getting closer to the answer that will turn out.

On page 77, which is probably Tuesday, December 8th, where the solution is fairly clear, I mean, the comment, “It’s clear, looking back, that mercaptoethanol oxidation plus coupling — plus coupling explains many of the results. However, also clear that at higher pH coupling is faster, as expected, since the reaction is approximately as follows.” And there is the scheme that became important. That’s a pro‑‑ [HSS-protein-SS-protein?] [00:16:00] plus mercaptoethanol leads to a split protein, with one SH group — one of the cysteines being as S– and the other as being [mercaptoethanol-SS?]. And then, in the presence of more [mercaptoethanol, SS?] — both cysteines in the protein are converted so SS‑ME, ME‑SS, which is clearly a stable product. And it says, after saying what the reaction is, “It’s clear that the reaction will not go without mercaptoethanol. And presumably the initial step,” which I call reaction 1, etc., etc. “Therefore, keep [00:17:00] mercaptoethanol concentration low and SS‑ME high.” That is the answer, in fact, which was the solution to this type of process, of splitting and getting a stable monomer from the polymers of haptoglobin. Various tests of this idea coming along, then.

And back to the idea of perhaps whether Factor B might make any difference, on page 85, Friday, December 11. [00:18:00] It looks as if Factor B has worked on the oxidation, as shown by the iodine test “but the splitting was poor.” And a diagram below. “Since these will interact, best to do” a complete test, where you don’t just do across or down a matrix but you do all cha‑‑ areas — an idea that Charlie Cotterman taught me, in Wisconsin, that it’s very important, in — when there are two variables that are var‑‑ that are being altered, and that each can affect the result, that one doesn’t just look at one horizontal row and one vertical row [00:19:00] but always complete the whole matrix — which is what is being stated, indirectly, at the bottom of this page, 85. Page 87, Factor B being looked at again. (pause)

So a summary, on page 91, the effect of temperature, effect of pH, the effect of mercaptoethanol concentration and Factor B — s‑‑ with a “future plan to prepare 50mgs of the monomer by using [00:20:00] 0.1M dithiodiglycol and 4‑mM mercaptoethanol, aerobically,” and add Factor B, 1 micromolar, at the end. I’m still thinking of oxidation in the resynthesis. So haven’t quite got the complete idea, of rever‑‑ of reversibility without thinking about oxidation. And some planned two-dimensional gels, on 93, with the run of the gel and the result, on the following pages — to try to test what’s happening.

Page [00:21:00] 99 goes to trypsin, antitrypsin tests — for no obvious reason — where serum plus trypsin — etc. And some tests on a emphysema patient planned, on Tuesday, December 22nd. Some trouble with electroosmosis. These were agar gels, 1.5% agar. And it was going to be a [Lorell?] test as an antibody [test?], on gel. [00:22:00] More work on trypsin, on December 22nd, continuing, with no clear motivation for these experiments being in the lab notebook.

[00:23:00] Still continuing with this antitrypsin, Monday, December 28th. This is — I can see now what it is. This is an attempt to find which band is antitrypsin. Page 120 makes this clear now, that the “Results of antitrypsin stain too poor to photograph.” But I’m trying to find which protein in serum is an antitrypsin protein. So that’s the motivation of the antitrypsin [00:24:00] experiments, which are — was not clear earlier on in the book. Some different assays of antitrypsin activity discussed, on Tuesday, December 29th, on page 123, a method of allying a variant and BAPA, whatever that is, and trypsin. (pause) [00:25:00] And although, on Wednesday, December 30th, and Thursday, December 31st, page 125, I do have the comment that “The results are very reproducible and tryps‑‑ and trypsin and substrate show excellent stability,” etc., etc. And the homozygote is about twenty‑fi‑‑ is about 20% of… So that this was a test of antitrypsin, with the gel.

Back to haptoglobin subunits, favorite topic, on January 7th, page 127, with thiodiglycol now being very clearly important. [00:26:00] So the degree of removal of the polymers is quite clear, although not commented on particularly on this page. Continuing with the thought and thinking also of dithiodiglycol with urea or with acetone. And first trials of resynthesis, on page 129. So now I’m feeling secure enough to try to think about resynthesis but probably being sidetracked a little — but the acetone or the urea tests — but made up various solutions [00:27:00] for use in this sort of experiment. And how it was set up, in the following pages. And then the result, on page 133, which shows a gel containing the water and the urea and the acetone tests and what is happening. But not very [in trouble?], as far as resynthesis is concerned.

Continuing, on Monday, January 11th, page 135, haptoglobin resynthesis tries — with none of the “very promising results” [00:28:00] statements. So must not have been particularly impressed. Thinking about storage, on page 137, further storage effects. Mak‑‑ rather a not very wise diversion but nonetheless there. Decided to allow some of the reaction to occur aerobically at the last stages — but in trying to get resynthesis of stored material. Another Bence Jones protein turned up, on page 139, Snyder Bence Jones protein.

Making the [bolt?] monomer of [00:29:00] haptoglobin 2‑2, a promising step, on Tuesday, January 12. “55mgs of Hp 2‑2 dissolved in,” etc, “washed,” etc., and exposed to 0.2-molar — or mixed with poi‑‑ equal volumes of a solution with 0.2-molar dithiodiglycol, 0.008-molar mercaptoethanol, final concentrations being 0.1-molar DTDG and 0.004 ME — that — conditions which are later settled on. And very good dissociation of the polymers into something that looks like a stable monomer, which can still bind hemoglobin. And, “Yield, [00:30:00] 51mgs.”

Future plans considered, on page 145. With ur‑‑ with and without urea, again, a bit of a diversion, on page 147. And here, on page 145, are various gels, being used to test with the ure‑‑ the urea — in fact, 0‑ up to 8-molar urea. (pause) With, interestingly, the writing on this page, again, here — two-thirds of the gels are written down me and one [00:31:00] gel neatly written down by, presumably, Nancy. The results of the gels are not terribly good. But they’re not commented on, anyway.

Trying to think of slow oxidation of the 0.002-molar monomer. Very little change since the initial 48‑hour –” [testing the?] columns. So, “Therefore, persist with anaerobic tests” — a good decision. Two-dimensional checks, on Saturday, June [sic] 16th, with some quite nice gels, showing the monomer [00:32:00] and the polymers obtained in these various experiments. And that ends this book.